The ability to silence genes via RNA interference (RNAi) was reported by Mello and Fire in 1998 (Fire et al., Nature (1998) 391:806-811). Since then, scientists have rushed to take advantage of the enormous therapeutic potential driven by targeted gene knockdown. This is evidenced by the fact that the first report of small interfering RNA (siRNA) mediated RNAi in human beings was reported only twelve years after the phenomenon was described in Caenorhabditis elegans (Davis et al., Nature (2010) 464:1067-1070). Unfortunately, this report represents the only successful therapeutic application to date. It is well understood that development of genetic drugs is slowed by the inability to deliver nucleic acids effectively in vivo. When unprotected, genetic material injected into the bloodstream can be degraded by DNAases and RNAases, or, if not degraded, the genetic material can stimulate an immune response (see, e.g., Whitehead et al., Nature Reviews Drug Discovery (2009) 8:129-138; Robbins et al., Oligonucleotides (2009) 19:89-102). Finally, in-tact siRNA must enter the cytosol, where the antisense strand is incorporated into the RNA-induced silencing complex (RISC) (Whitehead in supra). RISC associates with and degrades complementary mRNA sequences; this prevents translation of the target mRNA into protein, “silencing” the gene.
To overcome these barriers, nucleotides have been complexed with a wide variety of delivery systems, including polymers, lipids, inorganic nanoparticles and viruses (see, e.g., Peer et al. Nature Nanotechnology, (2007) 2:751-760). Although it had been used in commercial products for over thirty years, the cationic polymer polyethyleneimine (PEI) was first reported as an effective gene therapy delivery material in 1995 (Boussif et al., Proceedings of the National Academy of Sciences of the United States of America (1995) 92: 7297-7301). The amino groups of PEI are known to be protonated at physiological pH, facilitating electrostatic interactions with the negative phosphate backbone of the nucleotide (see, e.g., Suh et al., Bioorganic Chemistry (1994) 22:318-327). Branched PEI (BPEI) is one of the most highly characterized polymeric vectors for genetic delivery owing to increased interactions between the branched cation and the relatively linear phosphate backbone (Von Harpe et al., Journal of Controlled Release (2000) 69: 309-322). In addition to this stability, BPEI is considered an ideal delivery vector because the primary, secondary and tertiary amino groups have different pKas, and can therefore accept a large number of protons in the endosome without acidifying the membrane. Since acidification is required for nuclease activity that would disassemble the nucleotide, the nucleotide is protected from degradation. Furthermore, the influx of protons creates a gradient down which ions and water flow, an effect also known as “the proton sponge”. The endosomal membrane is filled like a balloon until it bursts open, releasing genetic material into the cytoplasm. In this way, BPEI is able to effectively ferry nucleic acids from the extracellular space into the cytoplasm (see, e.g., Akinc et al., Journal of Gene Medicine (2005) 7: 657-663).
The potential for effective delivery of nucleic acids with PEI has driven significant interest in the polymer (see, e.g., Incani et al., Soft Matter (2010) 6:2124-2138; and Howard, Advanced Drug Delivery Reviews (2009) 61:710-720). However, many authors have reported that efficacy, molecular weight and toxicity increase together (see, e.g., Godbey et al., Journal Of Biomedical Materials Research (1999) 45: 268-275). For example, it has been reported that a number average molar mass (Mn) of 25,000 BPEI (BPEI25,000) was required for effective DNA delivery, while Mn 1200 and Mn 600 PEI failed to yield expression in any trial (Richards-Grayson et al., Pharmaceutical research (2006) 23:1868-1876). In that same report, BPEI800 DNA transfection was compared to Mn 22,000 linear PEI (LPEI22,000) and BPEI25,000, and the authors found that while transfection was only seen with the BPEI25,000 formulation, BPEI25,000 was also found to be more cytotoxic. Regardless, many researchers have utilized BPEI25,000 to delivery DNA and siRNA (see, e.g., Alshamsan et al., Biomaterials (2009) 31:1420-1428; Kim et al., Bioconj. Chem. (2006) 17:241-244; Kim et al., J. Controlled Release (2006) 129:107-116; Kwon et al., Bioconj. Chem. (2008) 19:920-927; Jiang et al., Biopolymers (2008) 89:635-642; Creusat et al., Bioconjugate chemistry (2010) 21:994-1002; Bajaj et al., Bioconjugate chemistry (2008) 19:1640-1651; Furgeson et al., Pharmaceutical research (2002) 19:382-390; and Furgeson et al., Bioconjugate Chem (2003)14:840-847). In a recent report, BPEI25,000 was conjugated by N-acylation of the PEI to lipid tails and formulated with siRNA targeting STAT3, a protein required for tumor progression (Alshamsan et al. Biomaterials (2010) 31:1420-1428). In vitro analysis showed that the BPEI25,000-C16 conjugate reduced target gene expression by 50% at 25 nM in vitro and reduced tumor size by 50% at 0.3 mg/kg after intra-tumoral injections.
Investigators have sought to abrogate the cytotoxic effects of using BPEI25,000 in a variety of ways. For example, some investigators have conjugated lower molecular weight PEI segments together via biodegradable bonds in an effort to provide a new polymer which delivers nucleotides effectively like a BPEI25,000, but degrades into non-toxic constituents (see, e.g., Tarcha et al., Biomaterials (2007) 28:3731-3740; Breunig et al., Journal of Controlled Release (2008) 130:57-63; and Breunig et al., Proceedings of the National Academy of Sciences of the United States of America (2007) 104:14454-14459). Others have used BPEI2000 and BPEI1800 to a variety of hydrophobic chemical groups in an effort to bolster the molecular weight of the PEI polymer to provide a new polymer without the cytotoxic effects inherent to BPEI25,000. In one such report, lipid moieties were conjugated by N-acylation to BPEI2000 (2 kDa PEI) and evaluated for gene delivery (Neamnark, et al. Molecular Pharmaceutics (2009) 6:1798-1815). The authors reported that lipid conjugation improved gene delivery, but doses nearing 100 nM were not effective at transfecting the plasmid. In another report, BPEI1800 was conjugated to cholesterol in a molar ratio of cholesterol:BPEI1800 of approximately 1, but the authors required 5-15 μM for in vitro knockdown, indicating poor efficacy (see Kim et al., J. Controlled Release (2007) 118:357-363). Still others have investigated poly-siRNA conjugated to BPEI1800 for gene silencing (see, e.g., Lee et al., J. Controlled Release (2010) 141:339-346). However, the prevailing sense in the research community is that use of LPEI for the delivery of siRNA is still not as effective as use of BPEI, and that low molecular weight LPEI and BPEI polymers, i.e., having a number average molar mass (Mn) of ≤2000 (i.e., approximately ≤2 kDa), are inefficient materials for siRNA delivery (see, e.g., Boussif et al., Proceedings of the National Academy of Sciences of the United States of America (1995) 92: 7297-7301; and Philipp et al., Bioconjugate Chem. (2009) 20:2055-2061).
As described above, PEI mediated polynucleotide delivery has been well-studied, but, to date, no highly effective in vivo PEI-based delivery system has been reported. Thus, there continues to remain a need to develop a PEI-based polynucleotide delivery system which is as efficient a delivery system as a high molecular weight PEI, but with little to no cytotoxicity.